Bone is a complex mineralizing system composed of an inorganic or mineral phase, an organic matrix phase, and water. The inorganic mineral phase is composed of crystalline calcium phosphate salts while the organic matrix phase consists mostly of collagen and other noncollagenous proteins. Calcification of bone depends on the close association between the organic and inorganic phases to produce a mineralized tissue.
The process of bone growth is regulated to meet both structural and mechanical stresses. The cells involved in the processes of bone formation, maintenance, and resorption are osteoblasts, osteocytes, and osteoclasts. Osteoblasts synthesize the organic matrix, osteoid, of bone which after calcium phosphate crystal growth and collagen assembly becomes mineralized. Osteocytes regulate the flux of calcium and phosphate between the bone mineral and the extracellular fluid. Osteoclasts functions to resorb bone and are essential in the continuous process of bone remodelling. Disturbing the natural balance of bone formation and resorption leads to various bone disorders. Increased osteoclast activity has been demonstrated to lead to bone disease characterized by a decrease in bone density such as that seen in osteoporosis, osteitis fibrosa and in Paget's disease. All of these diseases are a result of increased bone resorption.
In order to understand the mechanisms involved which regulate bone cell functioning, it is important to be able to assess the normal function of bone cells and also the degree of perturbation of this activity in various bone diseases. This will lead to the identification of drugs targeted to restore abnormal bone cell activity back to within normal levels.
Several research groups have developed methods to directly observe the activity of isolated osteoclasts in vitro. Osteoclasts, isolated from bone marrow cell populations, have been cultured on thin slices of natural materials such as sperm whale dentine (Boyde et al Brit. Dent. J. 156, 216, 1984) or bone (Chambers et al J. Cell Sci. 66, 383, 1984). The latter group have been able to show that this resorptive activity is not possessed by other cells of the mononuclear phagocyte series (Chambers & Horton, Calcif Tissue Int. 36, 556, 1984). More recent attempts to use other cell culture techniques to study osteoclast lineage have still had to rely on the use of cortical bone slices (Amano et al. and Kerby et al J. Bone & Min. Res. 7(3)) for which the quantitation of resorptive activity relies upon either two dimensional analysis of resorption pit areas of variable depth or stereo mapping of the resorption volume. Such techniques provide at best an accuracy of approximately 50% when assessing resorption of relatively thick substrata. In addition these analysis techniques are also very time consuming and require highly specialized equipment and training. Furthermore, the preparation and subsequent examination of bone or dentine slices is neither an easy nor practical method for the assessment of osteoclast activity.
The use of artificial calcium phosphate preparations as substrata for osteoclast cultures has also met with little success. Jones et al (Anat. Embryol 170, 247, 1984) reported that osteoclasts resorb synthetic apatites in vitro but failed to provide experimental evidence to support this observation. Shimizu et al (Bone and Mineral 6, 261, 1989) have reported that isolated osteoclasts resorb only devitalized bone surfaces and not synthetic calcium hydroxyapatite. These results would indicate that functional osteoclasts are difficult to culture in vitro.
For these reasons it appears that the currently known methods used for culturing and measuring bone cell activity cannot provide consistent or reliable reference data for meaningful and statistical analysis. Therefore these methods are not suitable for wide scale screening for the assessment of bone cell function in normal health and/or in disease. These methods are also not suitable for diagnostic testing for disease.
Culturing systems developed for general tissue culture are also not suitable for the culturing of bone cells. U.S. Pat. No. 3,726,764 describes a microbiological chamber apparatus having a sealable sidewall access port for tissue culture directly onto a base for subsequent examination and storage. U.S. Pat. No. 3,745,091 describes a biological reaction chamber apparatus having a receptacle bonded to a base by a gasket for use in tissue culture. Neither of these systems is suitable or adaptable for the successful culture and assessment of both the activities of osteoclasts and osteoblasts.
The applicant's published PCT application (WO 94/26872) described a disc coated with a calcium phosphate based thin film which could be placed into individual containment wells onto which bone cells were cultured. Although this system was successful in culturing active bone cells, the system required a great deal of handling especially with respect to the cleaning and analysis of the individual discs. There was also the problem of bone cell migration from the discs as well as some variation in the film coating present on each disc from well to well. It was apparent that this system was not suitable for a large scale screening system aimed to quantitatively assess bone cell activity and diagnose bone cell disease.
We provide in the present invention a novel and improved bone cell culture device used to assess bone cell activity. The development of this bone cell culture device overcomes the difficulties previously encountered in the culturing of functional bone cells and the reliable assessment of bone cell activity.